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Abstract

Abstract We tested the effects of a National Cholesterol Education Program (NCEP) Step 2 diet (30% of calories or less as total fat, less than 7% saturated fat, and less than 200 mg cholesterol per day) on plasma lipid levels in normocholesterolemic and hypercholesterolemic middle-aged and elderly men and women. Thirty-two subjects were studied. Eight normolipidemic subjects (6 men and 2 women, mean age 56±13 years) with LDL cholesterol levels of less than 4.14 mmol/L (160 mg/dL) were given a baseline diet similar in composition to the diet currently consumed in the United States (35% of calories as total fat and 14% as saturated fat, with 147 mg cholesterol per 1000 kcal) for 6 weeks. Subjects were then placed on an NCEP Step 2 diet (26% total fat, 4% saturated fat, 45 mg cholesterol per 1000 kcal) for 24 weeks. In addition, 24 subjects (12 men and 12 women, mean age 62±12 years) with moderate hypercholesterolemia (LDL cholesterol levels of 4.14 mmol/L or above) were given a baseline diet for 6 weeks and then the NCEP Step 2 diet for 6 weeks. Energy intakes were adjusted to keep body weight constant throughout the study. In both normolipidemic and hypercholesterolemic subjects, consumption of the NCEP Step 2 diet was associated with significant changes in levels of total cholesterol (−20% and −16%, respectively), LDL cholesterol (−21% and −18%, respectively), and HDL cholesterol (−16% and −15%, respectively). The plasma total cholesterol/HDL cholesterol ratio and plasma triglyceride, VLDL cholesterol, and lipoprotein(a) levels were not affected by the diet in either group. Similar responses were seen in men and women. The range of diet responsiveness in the 32 subjects was very large, with LDL cholesterol changes ranging from +5% to −40%. Thus, an NCEP Step 2 diet is effective in significantly reducing total and LDL cholesterol levels without changing the total cholesterol/HDL cholesterol ratio under controlled isoweight conditions in middle-aged and elderly normolipidemic and hypercholesterolemic men and women. The wide individual variation in diet responsiveness may indicate genetic variability.

Presented, in part, at the annual meeting of the Federation of American Societies for Experimental Biology, Anaheim, Calif, April 26, 1994.

Received January 31, 1995.

Accepted May 8, 1995.

The National Cholesterol Education Program (NCEP) Expert Panel has recommended the consumption of the NCEP Step 2 diet as the strictest diet in the dietary treatment of hypercholesterolemia. This diet, restricted in total fat (30% or less of total calories), saturated fat (less than 7% of calories), and cholesterol (less than 200 mg/d) and with less than 15% of calories as monounsaturated fat and less than 10% of calories as polyunsaturated fat, is recommended for the reduction of plasma total and LDL cholesterol levels.1 These recommendations are justified by animal studies indicating that diets high in saturated fat and cholesterol induce atherosclerosis,2 studies documenting that populations consuming diets high in saturated fat and cholesterol have higher levels of LDL cholesterol and higher age-adjusted rates of coronary heart disease,3 and prospective dietary intervention studies demonstrating coronary heart disease risk reduction with restriction of dietary saturated fat and cholesterol.145678

The response to dietary restriction of fat, saturated fat, and cholesterol in both normolipidemic and hypercholesterolemic individuals has great clinical and public health relevance. Approximately half of the men and women in the Framingham Offspring Study have plasma LDL cholesterol concentrations above the level defined by the NCEP panel as desirable (3.36 mmol/L, or 130 mg/dL), and 24% of men and 22% of women have plasma LDL cholesterol levels above 4.14 mmol/L (160 mg/dL), the level defined as high risk.9 It is estimated that approximately 52 million Americans are candidates for dietary treatment of hypercholesterolemia.10 Moreover, if in subjects eligible for diet treatment there could be a 15% reduction in LDL cholesterol instead of a 5% reduction, the number of subjects eligible for drug therapy could be reduced by 50%.10 Therefore, dietary restriction of saturated fat and cholesterol may be critical in the general population. However, few studies have evaluated the efficacy of diets similar to the NCEP Step 2 diet in reducing plasma lipid levels in a controlled fashion.111213 There is a need for more information on the effects of NCEP Step 2 diets on plasma lipid levels.

The purpose of the present study was to determine the effects of a diet meeting NCEP Step 2 criteria on plasma lipid levels in middle-aged and elderly normolipidemic and hypercholesterolemic men and women under controlled isocaloric metabolic conditions. Our data indicate that, by complying with an NCEP Step 2 diet, both normolipidemic and hypercholesterolemic subjects may achieve significant reductions in plasma LDL cholesterol levels without significant changes in the total cholesterol/HDL cholesterol ratio. However, our data also indicate a significant biological variability in response to this diet in terms of LDL and HDL cholesterol lowering.

Methods

Study Subjects

All subjects were more than 40 years old and were not taking medications known to affect lipoprotein metabolism (eg, cholestyramine, colestipol, niacin, clofibrate, gemfibrozil, probucol, hydroxymethylglutaryl–coenzyme A reductase inhibitors, β-blockers, thiazide diuretics, diphenylhydantoin, cis-retinoic acid, ascorbic acid, estrogens, progestins, anabolic steroids, hydrocortisone, fish oil capsules, or thyroxin) at the time of screening or during the entire duration of the study. Female participants were all postmenopausal. Before enrollment in the study, subjects underwent a complete medical history and physical examination. None of the subjects had thyroid disease, diabetes mellitus, kidney disease, or liver disease by history or as assessed by laboratory tests (liver transaminases, alkaline phosphatase, glucose, creatinine, blood urea nitrogen, thyroid-stimulating hormone, T3, and T4). Subjects who smoked or consumed large amounts of alcohol were not enrolled in the study. A total of 32 subjects participated in this study. First, we studied eight normolipidemic subjects (six men and two women; mean age, 56±13 years) whose plasma LDL cholesterol levels at screening were less than 4.14 mmol/L (160 mg/dL) and whose other plasma lipid levels were normal (between the 10th and the 90th percentiles for age and sex) as defined by the Lipid Research Clinics data.14 These subjects consumed a baseline diet for 6 weeks and an NCEP Step 2 diet for 24 weeks. In addition, 24 subjects (12 men and 12 women; mean age, 62±12 years) with plasma LDL cholesterol levels at screening greater than or equal to 4.14 mm/L (160 mg/dL) were enrolled in the study; they consumed a baseline diet for 6 weeks and then an NCEP Step 2 diet for 6 weeks. A maximum of 4 weeks in the free-living state could separate the baseline and the NCEP Step 2 diet periods. The study protocol was approved by the Tufts University School of Medicine and New England Medical Center Human Investigation Review Committee. All subjects signed a consent form.

Diets

Meals for the baseline diet and the NCEP Step 2 diet were prepared by the Metabolic Research Unit of the USDA Human Nutrition Research Center on Aging at Tufts University and were provided to the participants. Meals consisted of breakfast, lunch, dinner, and one or two snacks, on a 3-day cycle menu. Subjects were requested to consume at least one meal per day at the Metabolic Research Unit on weekdays. Weekend meals were packaged and provided to the participants before the weekend. Blood pressure and body weight were measured at least three times per week. To keep each subject’s body weight constant (±1 kg) throughout the study, caloric intake was adjusted by means of proportional increments or decrements of all foods.

The composition of the baseline diet approximated that of the diet currently consumed in the United States15 ; it consisted of 35.4% of calories as fat and 14.1% of calories as saturated fat, and contained 147 mg cholesterol per 1000 kcal (Table 1⇓). The NCEP Step 2 diet consisted of 25.5% of calories as fat and 4.0% of calories as saturated fat, and contained 45 mg cholesterol per 1000 kcal. The difference in calories derived from total fat between the baseline diet and the Step 2 diet was compensated for by an increase in calories derived from carbohydrates. Triplicate preparations of each complete 3-day meal cycle for each of the two diets were analyzed for nutrient composition by Hazleton Laboratories America Inc. The protein, carbohydrate, fat, fatty acid, and cholesterol contents of these diets, as derived from chemical analysis, are provided in Table 1⇓. The total fiber content was calculated by use of food composition tables (grand database, release 867, USDA–Grand Forks Human Nutrition Research Center) and was 12.1 g/1000 kcal in the baseline diet and 16.6 g/1000 kcal in the NCEP Step 2 diet. Typical menus for the baseline and the NCEP Step 2 diets are shown in Table 2⇓.

Laboratory Measurements

In normocholesterolemic subjects, blood samples for plasma lipid measurements were collected at weeks 4, 5, and 6 of the baseline diet period and at weeks 4, 8, 12, 16, 20, and 24 of the NCEP Step 2 diet period. In hypercholesterolemic subjects, blood was collected at weeks 4, 5, and 6 of both the baseline diet and the NCEP Step 2 diet. Blood was collected, after a 12-hour overnight fast, in tubes containing 0.15% EDTA and centrifuged at 2500 rpm for 20 minutes at 4°C to separate plasma. HDL cholesterol levels were measured in plasma after precipitation of apo B–containing lipoproteins by the dextran sulfate–MgCl2 method.16 HDL3 cholesterol levels were measured directly from total plasma by use of a modification of the method of Warnick et al.17 Instead of performing sequential precipitations, in which HDL3 particles are isolated from the total HDL supernatants, we performed parallel precipitations directly from plasma. For the HDL3 precipitation, the concentrations of dextran sulfate and MgCl2 were adjusted to provide the same final concentrations as in the original method by addition of a 10% volume of precipitating reagent containing 19.1 g/L dextran sulfate and 1.95 mol/L MgCl2 to coprecipitate apo B–containing and HDL2 lipoproteins. After incubation at room temperature for 10 minutes, samples were centrifuged at 3500 rpm for 30 minutes at 4°C and the clear supernatants were used for cholesterol analysis. HDL2 cholesterol levels were calculated as the difference between HDL cholesterol and HDL3 cholesterol levels. Plasma was centrifuged at 4°C in a Beckman 50.3 Ti rotor at 39 000 rpm for 18 hours at a density of 1.006 g/mL to isolate VLDL, according to the Lipid Research Clinics methodology.18 Plasma total cholesterol and triglyceride levels, 1.006 g/mL infranatant cholesterol, and HDL cholesterol and HDL3 cholesterol levels were measured by automated enzymatic techniques with an Abbott Diagnostics Spectrum CCX bichromatic analyzer and Abbott enzymatic reagents.19 VLDL and LDL cholesterol concentrations were calculated as follows: VLDL cholesterol=total cholesterol−1.006 g/mL infranatant cholesterol, and LDL cholesterol=1.006 g/mL infranatant cholesterol−HDL cholesterol.

Our lipid methods are standardized through the Centers for Disease Control and Prevention (CDC) National Heart, Lung, and Blood Institute Lipid Standardization Program, and our laboratory serves as a member of the CDC Cholesterol Reference Method Laboratory Network.

In normolipidemic subjects, plasma apo A-I and apo B concentrations were measured in total plasma by noncompetitive enzyme-linked immunosorbent assays (ELISAs) with immunopurified polyclonal antibodies as previously described,2021 and plasma Lp(a) was measured with a commercially available ELISA (Strategic Diagnostics Inc) as previously described.22 In hypercholesterolemic subjects, plasma apo A-I, apo B, and Lp(a) concentrations were measured by immunoturbidimetric assays (IncStar). Coefficients of variation for all lipid assays were less than 5%, and for apolipoprotein assays they were less than 8%.

LDL particle size was determined by gradient gel electrophoresis on 2% to 16% nondenaturing polyacrylamide gels.23 The LDL particle score of each subject was calculated taking into account the relative area under the peak of the major and all minor LDL bands, as previously indicated.24 A higher LDL particle score represents a smaller LDL particle size.

Apo A-IV and apo E isoform phenotyping was performed on plasma obtained from all subjects by immunoblotting and isoelectric focusing methods, respectively, as previously described.2526

Statistical Analyses

The sas statistical program (SAS Institute) was used to perform all statistical analyses. proc glm was used to test by ANOVA for differences in mean values at weeks 4, 5, and 6 of the 6-week diet period and at weeks 4, 8, 12, 16, 20, and 24 of the 24-week diet period. Because no difference among time points within each dietary phase was observed, mean values for each of the dietary phases were calculated. A paired Student’s t test was carried out to test for differences between mean values during the NCEP Step 2 diet and the corresponding baseline value. Correlation analyses were performed by use of the proc corr procedure.

Results

Characteristics and plasma lipid levels at screening in normolipidemic and hypercholesterolemic subjects participating in the study are shown in Table 3⇓.

The effects of the NCEP Step 2 diet, compared with the baseline diet, on plasma lipid and apolipoprotein levels in normolipidemic and hypercholesterolemic subjects are shown in Table 4⇓. Stabilization of plasma lipid levels during the NCEP Step 2 diet, under isocaloric conditions, occurred by week 4 in both groups of subjects (data not shown). During consumption of the NCEP Step 2 diet, plasma total cholesterol, LDL cholesterol, and HDL cholesterol levels changed significantly in both normolipidemic subjects (–20%, –21%, and –16%, respectively) and hypercholesterolemic subjects (–16%, –18%, and –15%, respectively). The decrease in plasma HDL cholesterol levels observed during the NCEP Step 2 diet was due to decreases in both HDL2 and HDL3 cholesterol levels in both groups of subjects. As a result of similar decreases in total cholesterol and HDL cholesterol levels, a small and nonsignificant decrease in the total cholesterol/HDL cholesterol ratio was observed in both groups. The decreases in plasma apo B and apo A-I levels paralleled those in LDL and HDL cholesterol levels, respectively (Table 4⇓). Levels of plasma triglyceride, VLDL cholesterol, and Lp(a) were not significantly affected by the diet in these subjects (Table 4⇓).

Effects of a National Cholesterol Education Program Step 2 Diet on Plasma Lipid Levels in Normolipidemic and Hypercholesterolemic Subjects

LDL particle size was also measured at the ends of the baseline and the NCEP Step 2 diet periods (Table 4⇑). Consumption of the NCEP Step 2 diet was associated with an increase in LDL particle score, reflecting a decrease in LDL particle size in both groups of subjects. However, the diet-associated decrease in LDL size reached statistical significance only in the hypercholesterolemic group.

The individual LDL cholesterol and HDL cholesterol responses to diet in normolipidemic and hypercholesterolemic subjects participating in our study are shown in Fig 1⇓. The percent LDL cholesterol and HDL cholesterol change in response to the NCEP Step 2 diet in these subjects is shown in Fig 2⇓. A greater LDL cholesterol response to the diet in men than in women (−20.7% and −13.4%, respectively; P<.05) was observed. The percent HDL cholesterol response to the diet was similar in men and women (−14.5% and −15.3%, respectively). During the NCEP Step 2 diet, subjects with the apo E3/E4 phenotype (n=8) experienced a mean 18.6% decrease in LDL cholesterol levels compared with the baseline diet, whereas subjects with the E3/E3 phenotype (n=18) experienced a mean decrease of 15.2%. The difference in LDL cholesterol response to the diet in these apo E phenotype groups did not reach statistical significance. However, when analyses were performed separately for men and women, the LDL cholesterol response to the diet was significantly greater in men with the E3/E4 phenotype (n=5) than in those with the E3/E3 phenotype (n=10) (−28.7% and −14.9%, respectively; P<.03). LDL cholesterol response to the diet in women with the apo E3/E4 phenotype (n=3, −1.9%) was lower (P<.05) from that in women with the apo E3/E3 phenotype (n=8, −15.9%). However, this was a small sample size. Only three of the 32 subjects participating in our study had the apo A-IV-2 phenotype. This number was insufficient for statistical analysis. In univariate analyses, the diet-related percent change in LDL cholesterol levels was significantly associated with the percent change in HDL cholesterol levels (r=.41, P<.02). Body mass index was not associated with diet responsiveness in these subjects. In this study there was by design no effect on body weight.

Plots show percent changes in LDL cholesterol and HDL cholesterol levels in response to the National Cholesterol Education Program Step 2 diet in men (▪) and women (•) (n=32).

Discussion

Current guidelines of the NCEP Adult Treatment Panel indicate that some patients—those with plasma LDL cholesterol levels of 4.14 mmol/L (160 mg/dL) or above, those with LDL cholesterol above 3.36 mmol/L (130 mg/dL) and two or more risk factors for coronary heart disease, or those with LDL cholesterol levels of 2.59 mmol/L (100 mg/dL) or above in the presence of coronary heart disease—are to follow a Step 2 diet as the strictest dietary approach in the treatment of hypercholesterolemia.127 Several studies have determined the effects on plasma lipid levels of fat restriction or of the type of fat in the diet,282930313233 but very few have determined the effects of a diet similar to the NCEP Step 2 (30% total fat or less, less than 7% saturated fat, and less than 200 mg/d cholesterol) in a controlled fashion.111213 In a study by Grundy11 in which a liquid formula diet was used, plasma lipoprotein cholesterol levels in 11 moderately hypercholesterolemic men on a low-fat diet (20% fat: 6.7% saturated fat, 6.7% monounsaturated fat, and 6.7% polyunsaturated fat) were compared with levels in these subjects on a high-fat, high–saturated fat diet (40% fat: 18% to 25% saturated fat, 8% to 16% monounsaturated fat, and 4% to 7% polyunsaturated fat) and a high-fat, high–monounsaturated fat diet (40% fat: 4% saturated fat, 28% monounsaturated fat, and 8% polyunsaturated fat). Mean LDL cholesterol reductions were 15% on the low-fat diet and 21% on the high–monounsaturated fat diet, and reductions in HDL cholesterol levels were significantly greater on the low-fat diet than on the high–monounsaturated fat diet. In a recent study of 42 young to middle-aged normolipidemic men, plasma lipoprotein cholesterol levels on a high-fat diet (40% fat: 15% saturated fat, 14% monounsaturated fat, and 8% polyunsaturated fat and 189 mg cholesterol per 1000 kcal) were compared with values on a low-fat diet (19% fat: 4% saturated fat, 6.5% monounsaturated fat, and 5.2% polyunsaturated fat and 76 mg cholesterol per 1000 kcal) enriched in fiber.12 The low-fat diet resulted in decreases of 20% in both LDL and HDL cholesterol levels. We have recently reported the effects of NCEP Step 2 diets enriched with different types of vegetable oils (30% fat, 20% of calories derived from the selected type of oil) on plasma lipid levels of 15 middle-aged and elderly men and women.13 In these studies, decreases in LDL cholesterol levels were 13% to 17% and those in HDL cholesterol were 4% to 9%, compared with values on an average American diet, when subjects consumed diets rich in corn, canola, or olive oil. Therefore, despite differences in study design, study participant characteristics, and diet composition, the diet-induced changes in LDL and HDL cholesterol levels observed in our study are in overall agreement with those detected in other controlled studies.

As indicated in Table 2⇑, in our study the reduction in total and saturated fat in the NCEP Step 2 diet, compared with the average diet currently consumed in the United States, was achieved by excluding red meat, substituting low-fat dairy products for full-fat dairy products, and increasing the intake of fresh fruits, vegetables, and cereal-based products. These dietary changes are feasible and can be achieved by motivated individuals in the general population.

Within each population, plasma HDL levels have been inversely associated with the risk of coronary heart disease.3435 Some concern has been raised about the decrease in HDL cholesterol levels observed with low-fat diets. However, cross-cultural studies indicate that populations with very low intakes of fat in their diet have low rates of coronary heart disease despite low plasma HDL cholesterol levels.36 In addition, there are indications that, when these diets are consumed in a free-living state or when a regular program of physical exercise is associated with diet in overweight subjects, the reduction in HDL cholesterol may be minimized.37

In our subjects, there was a very slight and nonsignificant decrease in the total cholesterol/HDL cholesterol ratio. The decrease in plasma LDL cholesterol levels was paralleled by a similar decrease in apo B levels, suggesting a decrease in the number of circulating LDL particles. However, a change in the structural composition of LDL particles occurred as well, as suggested by a reduction in LDL particle size in both normal and hypercholesterolemic subjects during the consumption of the NCEP Step 2 diet. It has been shown that changes in plasma triglyceride levels are the strongest predictors of changes in LDL particle size.38 Because the consumption of the NCEP Step 2 diet was associated with decreases in both total cholesterol and LDL cholesterol levels without a change in triglyceride levels, it is likely that this relative increase in triglyceride levels was responsible for the LDL size change. Because there is a general concern about the atherogenic potential of small LDL, the change in LDL size that we observed in our subjects after consumption of the NCEP Step 2 diet deserves further attention.

The LDL cholesterol response to the NCEP Step 2 diet was greater in men with the E3/E4 phenotype than in men with the E3/E3 phenotype, in keeping with previous reports showing a greater response to dietary restriction in male subjects with apo E4.26

In this study, greater decreases in the HDL2 subfraction than in the HDL3 subfraction after dietary fat restriction were observed, in agreement with other studies.122939 It has been shown in kinetic studies that a reduction in dietary fat consumption is associated with decreases in apo A-I transport rates.40 Although the mechanism responsible for this phenomenon is not known at present, it is likely that the reduction in circulating apo A-I, without alteration in apo A-II concentrations, may lead to a preferential decrease in HDL particles containing apo A-I only, which are predominantly present in the HDL2 subfraction.41

Overall, our results indicate that an 18% decrease in LDL cholesterol may be achieved with only diet modification in hypercholesterolemic subjects. Recently the efficacy of the NCEP Step 2 diet in the reduction of LDL cholesterol levels in hypercholesterolemic patients has been questioned by a study reporting only a 5% decrease in LDL cholesterol.42 One problem with that study was that hypercholesterolemic subjects were selected who had previously been designated by their physicians to be candidates for drug therapy. Therefore, subjects less responsive to diet may have been selected. In addition, in outpatient diet studies, a major problem encountered by many investigators is the lack of patient compliance. Unlike the case in drug studies, a blinded design is not possible in diet studies, and investigators have relied on dietary questionnaires as instruments of compliance. However, it has been shown that the results of dietary questionnaires may have a high intraindividual variability.43 In addition, when diet records are used, patients may be more compliant with their diet during the recording days. In our study, all meals were provided to the participants, who were required to consume at least one meal every weekday at the Metabolic Research Unit, thus decreasing problems arising from lack of compliance. However, it is possible that the low content of saturated fat in our NCEP Step 2 diet (4%, when levels up to 7% are within the NCEP Step 2 guidelines) played a role in the marked LDL cholesterol–lowering response that we observed during that diet. It has been projected that if a 15% reduction in LDL cholesterol levels is achieved through dietary means in the US population, compared with only a 5% reduction, the number of subjects requiring drug treatment may be reduced by 50%.10

A final issue is the striking variability in individual response to the diet. In this study, under controlled conditions, we clearly documented a very high degree of variability of response to NCEP Step 2 diets with regard to both LDL and HDL cholesterol lowering. Katan and coworkers4445 observed similar variability in LDL cholesterol response to high-cholesterol diets. This large individual variability in the LDL cholesterol response to the diet indicates that genetic and environmental factors other than diet play a role in diet responsiveness.

Our data are consistent with a significant and beneficial effect on plasma LDL cholesterol levels of a diet low in total and saturated fat in both normocholesterolemic and hypercholesterolemic middle-aged and elderly men and women. Despite decreases in HDL cholesterol levels, no significant changes in the total cholesterol/HDL cholesterol ratio were observed.

Acknowledgments

This work was supported by the National Heart, Lung, and Blood Institute, National Institutes of Health, grant HL-39326, and by the US Department of Agriculture Research Service, contract 53-K06-5-10.

References

Expert Panel. Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). JAMA.1993;269:3015-3023.

Wood PD, Stefanick ML, Williams PT, Haskell WL. The effects on plasma lipoproteins of a prudent weight-reducing diet, with or without exercise, in overweight men and women. N Engl J Med.1991;325:461-466.